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Characterization of HIV-1 vpr nuclear import: analysis of signals and pathways.

Jenkins Y, McEntee M, Weis K, Greene WC - J. Cell Biol. (1998)

Bottom Line: Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy.Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites.Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Virology and Immunology, University of California, San Francisco, California 94141-9100, USA.

ABSTRACT
While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

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The two nuclear targeting signals of Vpr exhibit similar biological properties. (A–H) Nuclear import of IBB–βgal, Vpr(1–71)– βgal, Vpr(73–96)–βgal, and full-length Vpr–βgal in the presence of lysate (control) (A, C, E, and G) or with lysate containing 6 μM of  the RanQ69L mutant, which is unable to hydrolyze bound GTP (B, D, F, and H). (I–L) Nuclear import of IBB–βgal (I), Vpr(1–71)–βgal  (J), Vpr(73–96)–βgal (K), and Vpr–βgal (L) in the absence of added lysate. (M–P) Nuclear uptake of fluorescently labeled proteins  IBB–βgal (M), Vpr(1–71)–βgal (N), Vpr(73–96)–βgal (O), and Vpr–βgal (P) from a buffered solution 30 min into the import reaction.  In this study, cells were visualized without prior washing to evaluate the extent of nuclear accumulation. Images were photographed on  a Leitz diaplan fluorescence microscope illuminated by a mercury arc lamp. (Q–X) Import of IBB–βgal, Vpr(1–71)–βgal, Vpr(73–96)– βgal, and Vpr–βgal in the presence of lysate (control) (Q, S, U, and W) or lysate containing apyrase (2.5 U/ml) (R, T, V, and X) to deplete high-energy phosphates in the lysates. Lysates were preincubated with apyrase for 20 min at room temperature before their use in  the nuclear import assays.
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Figure 5: The two nuclear targeting signals of Vpr exhibit similar biological properties. (A–H) Nuclear import of IBB–βgal, Vpr(1–71)– βgal, Vpr(73–96)–βgal, and full-length Vpr–βgal in the presence of lysate (control) (A, C, E, and G) or with lysate containing 6 μM of the RanQ69L mutant, which is unable to hydrolyze bound GTP (B, D, F, and H). (I–L) Nuclear import of IBB–βgal (I), Vpr(1–71)–βgal (J), Vpr(73–96)–βgal (K), and Vpr–βgal (L) in the absence of added lysate. (M–P) Nuclear uptake of fluorescently labeled proteins IBB–βgal (M), Vpr(1–71)–βgal (N), Vpr(73–96)–βgal (O), and Vpr–βgal (P) from a buffered solution 30 min into the import reaction. In this study, cells were visualized without prior washing to evaluate the extent of nuclear accumulation. Images were photographed on a Leitz diaplan fluorescence microscope illuminated by a mercury arc lamp. (Q–X) Import of IBB–βgal, Vpr(1–71)–βgal, Vpr(73–96)– βgal, and Vpr–βgal in the presence of lysate (control) (Q, S, U, and W) or lysate containing apyrase (2.5 U/ml) (R, T, V, and X) to deplete high-energy phosphates in the lysates. Lysates were preincubated with apyrase for 20 min at room temperature before their use in the nuclear import assays.

Mentions: To further characterize the transport pathway(s) used by the two signals of Vpr, the requirement for GTP hydrolysis by Ran/TC4 was studied using a dominant-negative mutant of Ran, RanQ69L. This mutant binds GTP but is unable to hydrolyze this nucleotide triphosphate and consequently remains in the GTP-bound state. Addition of RanQ69L has been shown to block nuclear import of NLS- (Palacios et al., 1996) and M9-containing proteins (Nakielny et al., 1996) as well as nuclear import of U snRNPs (Palacios et al., 1996). Consistent with these results, inclusion of the RanQ69L mutant blocked IBB–βgal import (Fig. 5, A vs. B). In contrast, the RanQ69L mutant displayed no inhibitory effects on nuclear import mediated by either Vpr(1–71)–βgal (Fig. 5, C vs. D), Vpr(73–96)– βgal (Fig. 5, E vs. F), or full-length Vpr–βgal (Fig. 5, G vs. H). These results suggest that nuclear import mediated by either Vpr signal proceeds independently of Ran-mediated GTP hydrolysis.


Characterization of HIV-1 vpr nuclear import: analysis of signals and pathways.

Jenkins Y, McEntee M, Weis K, Greene WC - J. Cell Biol. (1998)

The two nuclear targeting signals of Vpr exhibit similar biological properties. (A–H) Nuclear import of IBB–βgal, Vpr(1–71)– βgal, Vpr(73–96)–βgal, and full-length Vpr–βgal in the presence of lysate (control) (A, C, E, and G) or with lysate containing 6 μM of  the RanQ69L mutant, which is unable to hydrolyze bound GTP (B, D, F, and H). (I–L) Nuclear import of IBB–βgal (I), Vpr(1–71)–βgal  (J), Vpr(73–96)–βgal (K), and Vpr–βgal (L) in the absence of added lysate. (M–P) Nuclear uptake of fluorescently labeled proteins  IBB–βgal (M), Vpr(1–71)–βgal (N), Vpr(73–96)–βgal (O), and Vpr–βgal (P) from a buffered solution 30 min into the import reaction.  In this study, cells were visualized without prior washing to evaluate the extent of nuclear accumulation. Images were photographed on  a Leitz diaplan fluorescence microscope illuminated by a mercury arc lamp. (Q–X) Import of IBB–βgal, Vpr(1–71)–βgal, Vpr(73–96)– βgal, and Vpr–βgal in the presence of lysate (control) (Q, S, U, and W) or lysate containing apyrase (2.5 U/ml) (R, T, V, and X) to deplete high-energy phosphates in the lysates. Lysates were preincubated with apyrase for 20 min at room temperature before their use in  the nuclear import assays.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132945&req=5

Figure 5: The two nuclear targeting signals of Vpr exhibit similar biological properties. (A–H) Nuclear import of IBB–βgal, Vpr(1–71)– βgal, Vpr(73–96)–βgal, and full-length Vpr–βgal in the presence of lysate (control) (A, C, E, and G) or with lysate containing 6 μM of the RanQ69L mutant, which is unable to hydrolyze bound GTP (B, D, F, and H). (I–L) Nuclear import of IBB–βgal (I), Vpr(1–71)–βgal (J), Vpr(73–96)–βgal (K), and Vpr–βgal (L) in the absence of added lysate. (M–P) Nuclear uptake of fluorescently labeled proteins IBB–βgal (M), Vpr(1–71)–βgal (N), Vpr(73–96)–βgal (O), and Vpr–βgal (P) from a buffered solution 30 min into the import reaction. In this study, cells were visualized without prior washing to evaluate the extent of nuclear accumulation. Images were photographed on a Leitz diaplan fluorescence microscope illuminated by a mercury arc lamp. (Q–X) Import of IBB–βgal, Vpr(1–71)–βgal, Vpr(73–96)– βgal, and Vpr–βgal in the presence of lysate (control) (Q, S, U, and W) or lysate containing apyrase (2.5 U/ml) (R, T, V, and X) to deplete high-energy phosphates in the lysates. Lysates were preincubated with apyrase for 20 min at room temperature before their use in the nuclear import assays.
Mentions: To further characterize the transport pathway(s) used by the two signals of Vpr, the requirement for GTP hydrolysis by Ran/TC4 was studied using a dominant-negative mutant of Ran, RanQ69L. This mutant binds GTP but is unable to hydrolyze this nucleotide triphosphate and consequently remains in the GTP-bound state. Addition of RanQ69L has been shown to block nuclear import of NLS- (Palacios et al., 1996) and M9-containing proteins (Nakielny et al., 1996) as well as nuclear import of U snRNPs (Palacios et al., 1996). Consistent with these results, inclusion of the RanQ69L mutant blocked IBB–βgal import (Fig. 5, A vs. B). In contrast, the RanQ69L mutant displayed no inhibitory effects on nuclear import mediated by either Vpr(1–71)–βgal (Fig. 5, C vs. D), Vpr(73–96)– βgal (Fig. 5, E vs. F), or full-length Vpr–βgal (Fig. 5, G vs. H). These results suggest that nuclear import mediated by either Vpr signal proceeds independently of Ran-mediated GTP hydrolysis.

Bottom Line: Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy.Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites.Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Virology and Immunology, University of California, San Francisco, California 94141-9100, USA.

ABSTRACT
While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

Show MeSH
Related in: MedlinePlus